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ANTICANCER RESEARCH 30: 1707-1712 (2010)

CEA-, Her2/neu-, BCRP- and Hsp27-positive Microparticles in Patients*

SUSANNE LIEBHARDT1, NINA DITSCH1, RIENK NIEUWLAND2, ANDREAS RANK3, UDO JESCHKE4, FRANZ VON KOCH5, KLAUS FRIESE1 and BETTINA TOTH6

1Department of Obstetrics and Gynecology - Großhadern, and 3Department of Internal Medicine III – Großhadern, Ludwig Maximilians University, 81377 Munich, Germany; 2Department of Clinical Chemistry, Academic Medical Center 1105 AZ Amsterdam, the Netherlands; 4Department of Obstetrics and Gynecology – Maistrasse, Ludwig Maximilians University, 80337 Munich, Germany; 5Department of Obstetrics and Gynecology, Klinikum Oritter Orden, 80638 Munich, Germany; 6Department of Gynecological Endocrinology and Reproductive Medicine, Ruprecht Karls University, 69115 Heidelberg, Germany

Abstract. Background: This is the first prospective case- So far, the only serum tumour markers for breast cancer are control study that evaluates the expression of tumour-specific cancer antigen (CA) 15-3 and carcinoembryonic antigen antigens on circulating microparticles (MP) in breast cancer (CEA). Neither marker is used in diagnosis of primary breast patients and in women with benign breast tumour. Materials cancer but only to diagnose recurrent disease and to evaluate and Methods: MP were determined by flow cytometry in response to chemotherapy (1). Prognostic factors for breast patients with breast cancer (n=34; T1 (n=19) and T2 (n=15)) cancer are the status of axillary lymph nodes, age, tumour and women with benign breast tumour (n=19). Results: load and histological parameters (2), urokinase-type Patients with lymph node metastases (N1, n=9) showed plasminogen activator (uPA) and its inhibitor (PAI-1) (3), significantly higher numbers of annexin V+ MP (p=0.042), and the presence of bone marrow micrometastases (4). CD66+ MP (p=0.045), BCRP1+ MP (breast cancer resistance Several investigators have demonstrated elevated ) (p=0.025) and Hsp27+ MP (p=0.034) than controls. concentrations of circulating microparticles (MPs) in breast Furthermore, T1 patients had significantly higher levels of cancer patients (5-7). MPs are cellular membrane vesicles of annexin V+ MP (p=0.004), CD66+ MP (p=0.025), BCRP1+ a size between 0.1 and 1.0 μm, which are shed into the MP (p=0.008) and Hsp27+ MP (p=0.02) than controls. plasma during cell activation and apoptosis. (8) MPs are Conclusion: Significant differences are present between breast present in the plasma of healthy humans, where they cancer patients with lymph node metastases and controls originate mainly from platelets, followed by endothelial concerning annexin V-, CD66-, BCRP1- and Hsp27-positive cells, granulocytes and erythrocytes. (9) Increased plasma MP. To specify the role of these MP subpopulations in breast concentrations of MPs have been reported in vascular cancer progression, further studies enrolling larger patient diseases, diabetes and severe trauma (10) and in several types groups are part of ongoing research. of cancer including gastric (11), lung (12), and (13), leukaemia (14), myeloproliferative disorders (15), pancreatic (5) and breast cancer (5-7). In patients with , high platelet-derived MP (PMP) is correlated *This work contains parts of the doctoral theses of Kerstin Steinig with short overall survival. (16) MP promote tumour growth, and Susanne Liebhardt. spreading and tumour-induced neovascularization (11). There are several antigens which have been identified on Correspondence to: Susanne Liebhardt, Department of Obstetrics breast cancer tissue: cluster of differentiation 66 (CD66), and Gynecology – Großhadern, Ludwig Maximilians University, Marchioninistr. 15, D-81377 Munich, Germany. Tel: +49 8970952856, human epidermal growth factor receptor 2 (Her2/neu), breast Fax: +49 8970955854, e-mail: [email protected] cancer resistance protein (BRCP) and heat-shock protein 27 (Hsp27). CD66 is known as the CEA (carcinoembryonic Key Words: CEA, Her2/neu, BCRP, HSP27, microparticles. antigen) family of molecules (1) and plays a role in cell

0250-7005/2010 $2.00+.40 1707 ANTICANCER RESEARCH 30: 1707-1712 (2010) adhesion and rejection processes and therefore in tumour and IgG-PE were from Immuno Quality Products (Groningen, growth and spreading (1, 17). Elevated levels of CEA occur in Netherlands) and IgG-FITC from Immunotech (Marseille, France). patients with colorectal (18), but also in patients Anti-CD66-PE (detecting CD66c, e) and anti-Her2/neu-PE were from BD Biosciences Pharmingen (San Jose, USA), anti-BCRP1- with breast, lung, ovarian, prostate, hepatocellular and FITC from Chemicon International (Billerica, USA) and anti- (19). Her2/neu, a transmembrane Hsp27-FITC from Stressgen (Victoria, Canada). All and from the epidermal growth factor receptor family annexin V were diluted with phosphate-buffered saline (PBS; 154 (EGFR) (20, 21), is expressed by various epithelial tissues. mmol/l NaCl, 1.4 mmol/l phosphate, pH 7.4). Final dilutions were: Her2/neu is overexpressed in 10-31% of invasive breast annexin V-FITC 1:100 (v/v), annexin V-PE 1:200, IgG-PE 1:10, cancers, where it is a marker of poor prognosis (22-24). BCRP, IgG-FITC 1:10, anti-Her2/neu-PE 1:2, BCRP1-FITC 1:5, Hsp27- a 72 kDa plasma membrane protein from the ABC (ATP FITC 1:5, anti-CD66-PE was used undiluted. Isolation and identification of MP were performed as described binding cassette) transporter family (25), was first discovered in by Nieuwland (30). In brief, frozen plasma (250 μl) was thawed doxorubicin-resistant breast cancer cells (26). Endogenous slowly on melting ice for approximately one hour. After BCRP expression is found in placenta, liver, bowel, ovary, centrifugation at 17570×g and 20˚C for 30 minutes, 225 μl of MP- blood vessels and in the ductuli and lobuli of the breast (27). free supernatant was taken off. The remaining MP pellet was diluted Heat-shock mediate protein transport and folding and with 225 μl of PBS containing 10.9 mmol/l trisodium citrate are cytoprotective. (28) Hsp27 belongs to the family of small (PBS/citrate buffer), re-suspended and centrifuged again for 30 Hsps (15-30 kDa) (28). An overexpression of Hsp27 is found minutes at 17570×g and 20˚C. Afterwards the supernatant (225 μl) was removed, 75 μl of PBS/citrate buffer was added, and the MP in various like breast, ovarian, colorectal, pellet was resuspended again. Five μl of the MP suspension was hepatocellular, kidney, lung and prostate carcinoma (29). diluted in 35 μl CaCl2 (2.5 mmol/l)-containing PBS. For MP The aim of our prospective case–control study was to staining, 5 μl APC-labelled annexin V and 5 μl of a cell-specific detect tumour-specific MPs in women with breast cancer. monoclonal or isotype-matched control antibodies were Therefore, MP-expressing CD66, Her2/neu, BCRP and added to the specimen. For detection of tumour-specific MP, Hsp27 were evaluated in breast cancer patients using women antibodies against Her2/neu, BCRP, CD66 and Hsp27 were used. with benign breast tumours well as controls. Samples were incubated in the dark for 15 minutes at room temperature. The reaction in all samples was stopped with 900 μl calcium buffer (2.5 mmol/l), except for the annexin V control, where Patients and Methods citrate-containing PBS (900 μl) was added. MP were analysed in a FACScan flow cytometer (Becton Study population. Newly diagnosed patients with histologically Dickinson, Heidelberg, Germany) using Cell Quest Software (Becton proven breast cancer (n=34) were included in the study before Dickinson, San Jose, CA, USA). Forward scatter (FSC) and side surgical treatment or neoadjuvant chemotherapy and classified scatter (SSC) were set at a logarithmic gain. MPs were identified on according to the TNM system: 19 patients had tumours <2 cm (T1) basis of their size and density and their capacity to bind to a cell- and 15 had tumours of a size between 2 and 4 cm (T2). 23 patients specific and annexin V. Cell-specific labelling had negative axillary lymph nodes (N0), 9 had positive lymph nodes with monoclonal antibodies was corrected for identical concentrations (N1) and in 2 patients the nodal state was unknown (Nx). Women of isotype-matched control antibodies and annexin V measurements with a benign breast tumour (n=19) were included in the control were corrected for autofluorescence. The concentration of MPs per group before core biopsy of breast tumours which had been detected litre of plasma was estimated according to Berckmans (9). during routine mammography. Neither patients nor controls smoked or were taking oral contraceptives or hormonal treatment. Patients Statistics. Parametrically distributed data were expressed as with the confounding factors hypertension, history of cancer or mean±standard deviation (SD) (minimum-maximum). All other data thrombosis, aspirin or low molecular weight heparin (LMWH) were presented as the median (Q1-Q3=interquartile range). treatment were excluded. All patients were Caucasians. Signed Independent variables were analyzed by Mann-Whitney-U-test, all informed consent was obtained from all participants, allowing others by using Chi-square and Fisher’s exact test. P-values <0.05 analysis of all clinical and laboratory data mentioned in this paper. were regarded as statistically significant. Data were examined with The Human Investigation Review Board of the Ludwig Maximilians SPSS (SPSS, Chicago, IL, USA) 16.0 for Windows. University, Munich, Germany, approved the study. Results Blood sampling and measurements. All blood samples were collected between 09.00-12.00 am by a single experienced medical Study population. Patients and controls did not differ student by puncture of the antecubital vein without tourniquet significantly concerning age, body mass index, haemoglobin through a 20-gauge needle. level, platelet count, leucocyte count, CEA and CA15-3 For MP analysis, platelet-poor plasma was prepared by levels (Table I). centrifugation at 1550×g for 20 minutes within 15 minutes after collection. The plasma was then snap frozen in liquid nitrogen for 15 minutes and stored at –80˚C until assayed. Microparticle analysis. In brief, it was possible to detect + + Reagents for microparticle analysis. Fluorescein isothiocyanate different subpopulations of MPs (CD66 , Her2/neu , (FITC)-labelled annexin V, phycoerythrin (PE)-labelled annexin V, BCRP1+, Hsp27+ MP). The Hsp27+ subgroup represented

1708 Liebhardt et al: Tumour-specific Antigens on Circulating Microparticles in Breast Cancer

Table I. Study population.

Age (years) BMI (kg/m2) Hb (g/dl) Platelets (×106/l) Leukocytes (×106/l) CEA (ng/ml) CA15-3 (U/l)

Control 51.3±16.2 23.4±4.2 13.7±0.9 281.8±49.4 7.6±2.5 1.9±1.2 18.9±7.0 (n=19) (19-80) (18.6-30.8) (12.3-15.4) (199-366) (4.9-16.0) (0.7-.3.8) (7.5-27.7) Patients 59.2±13.8 25.4±3.8 13.3±1.2 281.9±61.6 6.7±1.3 4.1±10.6 18.6±9.4 (n=34) (26-83) (17.2-33.6) (11.3-15.7) (178-426) (4.5-9.0) (0.4-56.1) (5.6-58.0)

Demographic patient data are shown. Parametrically distributed data are presented as mean±standard deviation (minimum-maximum).

Table IIa. Annexin V+ MPs, CD66+ MPs, Her2/neu+ MPs, BCRP+ MPs and Hsp27+ MPs (×106/l) in the study population.

Annexin V CD66 Her-2/neu BCRP Hsp27

Control 5468 835 26 600 2344 (n=19) (1900-9834) (219-1507) (6-106) (251-1720) (608-5060) (4306-6624) (610-1066) (20-43) (453-715) (1763-3258) Patients 7296 998 30 728 2825 (n=34) (1717-17455) (367-2662) (10-229) (246-2012) (891-8447) (4316-7296) (732-1621) (19-67) (562-1299) (1869-4741) T1 9353 1167 34 769 3585 (n=19) (1717-17455) (506-2628) (10-207) (407-1399) (891-8447) (6563-11240) (848-1656) (22-90) (639-1301) (2702-5119) T2 5707 790 23 651 2267 (n=15) (1904-13643) (367-2662) (11-229) (246-2012) (1072-6227) (2565-8701) (606-1461) (16-52) (371-1280) (1572-3541)

Data are presented as median value, (minimum-maximum), (interquartile range).

Table IIb. Annexin V+ MPs, CD66+ MPs, Her2/neu+ MPs, BCRP+ MPs and Hsp27+ MPs (×106/l) in nodal-positive and nodal-negative breast cancer patients.

Annexin V CD66 Her-2/neu BCRP Hsp27

N0 7161 859 27 700 2634 (N=23) (1717-17455) (435-2662) (11-125) (329-2012) (891-8447) (2565-9787) (726-1534) (16-66) (433-997) (1665-2634) N1 8707 1301 34 796 3943 (n=9) (3191-14056) (367-2628) (10-229) (246-1797) (1626-6381) (5502-12565) (997-1453) (22-163) (689-1481) (2571-5486)

Data are presented as median value, (minimum-maximum), (interquartile range) in patients with positive (N1) or negative (N0) axillary lymph node metastases as compared to controls.

38.7% of the total number of MPs in patients and 42.9% Considering nodal status and MP count, significantly in controls. A total of 13.7% (patients) and 15.3% higher MP levels were found in patients with lymph node (controls) of annexin+ MPs were CD66+; 10.0% (patients) metastases (N1) compared to the control group (p=0.042) and 11.0% (controls) of annexin V+ MPs were BCRP+; (Table IIb, Figure 1). There were also significant differences 0.4% (patients) and 0.5% (controls) of annexin V+ MPs between N1 patients and controls with regard to CD66+ MPs were Her-2/neu+. (p=0.045), BCRP+ MPs (p=0.025) and Hsp27+ MPs Patients with smaller tumour size (T1) had significantly (p=0.034). The concentration of Her2/neu+ MPs was higher concentrations of annexin V+ MPs (p=0.004), elevated in N1 patients compared to controls without CD66+ MPs (p=0.025), BCRP+ MPs (p=0.008) and reaching significance (Table IIb, Figure 1). Hsp27+ MPs (p=0.02) than controls (Table IIa). However, Thus, the total number of MPs as well as three of the four MP levels did not differ significantly between T2 patients measured subpopulations were significantly elevated in N1 and controls. patients compared to controls. With regard to N1 patients, no

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Figure 1. a: Annexin V+ MPs in study population. Concentrations of annexin V+ MPs (x106/l) in the study population. Data presented as boxplot; p<0.05=significant. Y-axis: Concentration of annexin V+ MPs (×106/l), x-axis: patients with positive axillary lymph nodes (N1), negative axillary lymph nodes (N0) and women with benign breast tumour (benign). b: CD66+ MP in study population. Concentrations of CD66+ MP (×106/L) in the study population. Data presented as boxplot; p<0.05=significant. Y-axis: Concentration of CD66+ MPs (×106/l), x-axis: patients with positive axillary lymph nodes (N1), negative axillary lymph nodes (N0) and women with benign breast tumour (benign). c: Her2/neu+ MP in study population. Concentrations of Her2/neu+ MPs (×106/l) in the study population. Data presented as boxplot; p<0.05 = significant. Y-axis: Concentration of Her-2+ MPs (×106/l, x-axis: patients with positive axillary lymph nodes (N1), negative axillary lymph nodes (N0) and women with benign breast tumour (benign). d: BCRP+ MPs in study population. Concentrations of BCRP+ MPs (×106/l) in the study population. Data presented as boxplot; p<0.05 = significant. Y-axis: Concentration of BCRP1+ MPs (×106/l), x-axis: patients with positive axillary lymph nodes (N1), negative axillary lymph nodes (N0) and women with benign breast tumour (benign). e: Hsp27+ MPs in study population. Concentrations of Hsp27+ MPs (×106/l) in the study population. Data presented as boxplot; p=0.05=significant. Y-axis: Concentration of Hsp27+ MPs (×106/l), x-axis: patients with positive axillary lymph nodes (N1), negative axillary lymph nodes (N0) and women with benign breast tumour (benign).

1710 Liebhardt et al: Tumour-specific Antigens on Circulating Microparticles in Breast Cancer significant differences were present concerning the Most prognostic parameters in breast cancer require concentrations of annexin V+ MPs as well as all extraction of tumour tissue, lymph nodes or bone marrow all subpopulations in comparison to N0 patients (Table IIb). of which are invasive methods. The detection of tumour markers in peripheral blood would be of value for patients Discussion and health care professionals. Attempts have been made to detect disseminated tumour cells in the peripheral blood This prospective case–control study is the first report addresing using several markers, among others CEA and Her2/neu the exposure of cancer-related markers on circulating MPs in (31). Our preliminary data suggest differences in the breast cancer patients and women with benign breast tumour. expression of cancer-related MP between breast cancer The total numbers of MPs as well as three of the four measured patients and controls. Future investigations on MP subpopulations (CD66+, BCRP+, Hsp27+) of MPs were concentrations in breast cancer patients should focus on elevated in patients with lymph node metastases compared to follow-up and survival rates. In particular, the possible role women with benign breast tumour. Since MPs from cancer of MPs as a prognostic factor should be highlighted in the cells have been implicated to contribute to tumour growth and presence of a larger patient group, including patients with spreading via various mechanism, elevated levels of circulating distant metastases. MP expressing cancer-related markers may reflect cancer activity in these patients. Acknowledgements Only few studies are currently reported concerning MP concentrations in breast cancer patients (5-7). In our former We gratefully acknowledge the following colleagues: Andrea Peichl and Marianne Fileki from the Großhadern Ludwig- studies, PMP, leucocyte-derived MP (LMP) and endothelial- Maximilians-University, Munich, Germany Department of cell-derived MP (EMP) were investigated by flow cytometry Obstetrics and Gynecology for technical assistance; Dr. Andreas and electron microscopy in breast cancer patients (6, 7) The Crispin from the Großhadern Ludwig Maximilians University, total number of MP as well as the number of PMP were Munich, Germany Department of Medical Informatics, Biometry significantly higher in patients than in controls, increasing and Epidemiology for support in statistical analyses; Dr. med with augmenting tumour size, and they were highest in Claudia Perlet from the Großhadern Ludwig-Maximilians- patients with distant metastases (6). In breast cancer patients, University, Munich, Germany Department of Radiology for help in acquisition of study patients. 82.3% of MP originated from platelets, 14.6% from activated Bettina Toth was supported by the Ludwig Maximilians endothelial cells and 0.3% from leucocytes (6). Furthermore, University excellent Mentoring program of the LMU, Munich, significant differences in LMP concentrations were present Germany. between breast cancer patients and controls, which may reflect the disease stage in these patients (7). The sensitivity- References specificity profile of LMP was equal to that of the established tumour marker CA15-3 (7). 1 Seregni E, Coli A and Mazzucca N: Circulating tumour markers Since MPs may expose surface proteins, indicating the cell in breast cancer. Eur J Nucl Med Mol Imaging 31(Suppl 1): S15- from which they originate (10), it is hypothesised that MP shed 22, 2004. 2 Engel J et al: The process of metastasisation for breast cancer. by cancer cells would be detectable in plasma samples from Eur J Cancer 39(12): 1794-1806, 2003. breast cancer patients by using specific antibodies directed 3 Look MP et al: Pooled analysis of prognostic impact of urokinase- against cancer- or epithelial-specific structures. Our present type plasminogen activator and its inhibitor PAI-1 in 8377 breast study shows significantly elevated concentrations of MP cancer patients. J Natl Cancer Inst 94(2): 116-128, 2002. expressing cancer-related antigens in patients with lymph node 4 Braun S et al: A pooled analysis of bone marrow micrometastasis metastases compared to controls. However, these markers are in breast cancer. N Engl J Med 353(8): 793-802, 2005. not specific for breast cancer but they are also expressed in 5 Tesselaar ME et al: Microparticle-associated activity: a link between cancer and thrombosis? J Thromb healthy breast tissue as well as in various other carcinomas. In Haemost 5(3): 520-527, 2007. particular, Hsp27 is expressed in various tissues, which may 6 Toth B et al: Platelet-derived microparticles and coagulation + explain the high percentage of Hsp27 MPs. activation in breast cancer patients. Thromb Haemost 100(4): MPs could possibly be of use as a prognostic factor. 663-669, 2008. Established prognostic factors in breast cancer are currently 7 Toth B et al: Circulating microparticles in breast cancer patients: the status of axillary lymph nodes, age and tumour load as a comparative analysis with established biomarkers. Anticancer well as histological parameters (2). New markers of breast Res 28(2A): 1107-1112, 2008. 8 VanWijk MJ et al: Microparticles in cardiovascular diseases. cancer are urokinase-type plasminogen activator (uPA) and Cardiovasc Res 59(2): 277-287, 2003. its inhibitor (PAI-1), which contribute to invasiveness and 9 Berckmans RJ et al: Cell-derived microparticles circulate in dissemination potential of tumour cells (3) as well as the healthy humans and support low grade thrombin generation. presence of bone marrow micrometastases (4). Thromb Haemost 85(4): 639-646, 2001.

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10 Martínez MC et al: Shed membrane microparticles from 22 Kaptain S, Tan LK and Chen B: Her-2/neu and Breast Cancer. circulating and vascular cells in regulating vascular function. Am Diagnost Mol Pathol 10(3): 139-152, 2001. J Physiol Heart Circ Physiol 288: 1004-1009, 2005. 23 Pauletti G et al: Detection and quantification of HER-2/neu gene 11 Kim CW et al: Extracellular membrane vesicles from tumor amplification in human breast cancer archival material using cells promote angiogenesis via sphingomyelin. Cancer Res fluorescence in situ hybridization. Oncogene 13: 63-72, 1996. 62(21): 6312-6317, 2002. 24 Slamon DJ et al: Human breast cancer: correlation of relapse 12 Kanazawa S et al: Monocyte-derived microparticles may be a and survival with amplification of the HER-2/neu oncogene. sign of vascular complication in patients with . Lung Science 235: 177-182, 1987. Cancer 39: 145-149, 2003. 25 Perez-Tomas R: Multidrug resistance: retrospect and prospects 13 Hron G et al: Tissue factor-positive microparticles: Cellular in anticancer drug treatment. Curr Med Chem 13(16): 1859- origin and association with coagulation activation in patients 1876, 2006. with colorectal cancer. Cellular Proteolysis and , 2007. 26 Ishikawa T et al: Transport mechanism-based drug molecular 14 Savasan S et al: Leukaemia/ cell microparticles in design: novel camptothecin analogues to circumvent ABCG2- childhood mature B cell neoplasms. J Clin Pathol 57(6): 651- associated drug resistance of human tumor cells. Curr Pharm 653, 2004. Des 12(3): 313-325, 2006. 15 Falanga A, Barbui T and Rickles FR: Hypercoagulability and 27 Ejendal KF and Hrycyna CA: Multidrug resistance and cancer: tissue factor gene up-regulation in hematologic malignancies. the role of the human ABC transporter ABCG2. Curr Protein Semin Thromb Hemost 34(2): 204-210, 2008. Pept Sci 3(5): 503-511, 2002. 16 Helley D et al: Platelet microparticles: a potential predictive 28 Concannon CG, Gorman AM and Samali A: On the role of factor of survival in hormone-refractory prostate cancer patients Hsp27 in regulating apoptosis. Apoptosis 8(1): 61-70, 2003. treated with docetaxel-based chemotherapy. Eur Urol, 2008. 29 Arrigo AP et al: Hsp27 (HspB1) and alphaB-crystallin (HspB5) 17 Egawa K et al: The carcinoembryonic antigen (CEA) family as therapeutic targets. FEBS Lett 581(19): 3665-3674, 2007. (CD66) expressed in melanocytic naevi is not expressed in blue 30 Nieuwland R et al: Cell-derived microparticles generated in naevuscell naevi in dendritic type. J Cutan Pathol 27(7): 351- patients during cardiopulmonary bypass are highly procoagulant. 358, 2000. Circulation 96(10): 3534-3541, 1997. 18 Stearns V, Yamauchi H and Hayes DF: Circulating tumor 31 Gilbey AM et al: The detection of circulating breast cancer cells markers in breast cancer: accepted utilities and novel prospects. in blood. J Clin Pathol 57(9): 903-911, 2004. Breast Cancer Res Treat 52(1-3): 239-259, 1998. 19 Cheung KL, Graves CR and Robertson JF: Tumour marker measurements in the diagnosis and monitoring of breast cancer. Cancer Treat Rev 26(2): 91-102, 2000. 20 Coussens L et al: Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230: 1132-1139, 1985. 21 Shih C et al: Transforming genes of carcinomas and Received August 19, 2009 introduced into mouse fibroblasts. Nature 290: Revised March 23, 2010 261-264, 1981. Accepted March 26, 2010

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